Project Summary Lung cancer is the leading cause of cancer-related deaths in the United States, and the 5-year survival rate of all lung cancer patients combined is only about 18%. The implementation of cancer immunotherapeutics for solid tumors such as lung cancers has shown great promise and provided the possibility for improved outcome in a small percentage of patients. However, the majority of patients show little to no response or acquire resistance during treatment with checkpoint inhibitors delivered as a monotherapy. Therefore, identifying resistance mechanisms and potential combination therapy approaches is a critical need to improve response rates to immune checkpoint inhibitors and patient prognosis. To address this, a clinically relevant in vivo shRNA dropout screen focused on genes encoding for FDA-approved drug targets (FDAome) was performed in epithelial and mesenchymal Kras/p53 (KP) mutant murine lung cancer cells. Mice were then treated with either isotype or anti- PD-1 antibody. Sequencing for the barcoded shRNAs revealed that Ntrk1 was significantly depleted from mesenchymal tumors challenged with PD-1 blockade compared to isotype treated tumors, suggesting it provides a survival advantage to these tumor cells when under immune system pressure. Preliminary data confirmed Ntrk1 transcript levels are upregulated in mesenchymal tumors treated with PD-1 inhibitors and cell lines derived from resistant tumors, and analysis of human NSCLC cell lines revealed that Ntrk1 mRNA levels correlate with a more aggressive, mesenchymal cell phenotype. Additionally, Ntrk1 overexpressing cells upregulate PD-L1 expression when co-cultured with splenocytes through upregulation of JAK signaling. Stable knockdown of Ntrk1 in mesenchymal murine KP mutant lung cancer cells reduced tumor growth in vivo and analysis of tumor- infiltrating T cell populations via flow cytometry showed that CD8+ T cell exhaustion was significantly reduced, whereas overexpression of Ntrk1 promoted CD8+ T cell exhaustion, thus decreasing effector status. These tumors also have an altered microenvironment, with upregulation of classically immunosuppressive cytokines such as IL-10. PD-1 protein levels were also significantly increased in Ntrk1-high human NSCLC cell lines, providing additional evidence that Ntrk1 may be a modulator of immune system functionality in human lung disease. The central hypothesis of the proposed work is that Ntrk1 upregulation causes acquired resistance to PD-1 blockade via aberrant JAK signaling and downstream CD8+ T cell dysfunction, thereby promoting tumor cell survival. A variety of powerful tools will be utilized to test this hypothesis, including time-lapse imaging of dynamic T cell-tumor cell interactions, genetically-engineered and syngeneic preclinical models of lung cancer to analyze immune subpopulations as a function of Ntrk1 expression, and IHC analyses of human NSCLC tissue samples. The goal of the proposed work is to provide strong evidence for a rational drug combination of Ntrk1 inhibitors with PD-1 blockade to be carried forward into the clinic to abrogate immune checkpoint blockade resistance and ultimately improve patient outcomes.